1. Field of Invention
The invention relates to the field of cell, tissue and organ preservation. More specifically, the invention relates to a method for treatment of cellular materials with sugars prior to preservation in an effort to enhance cell survival post-preservation. This is particularly important because the sugars, such as trehalose and sucrose, are not cytotoxic to cells and therefore may not have to be removed before living, natural or man-made cellular materials are transplanted into humans or animals.
2. Description of Related Art
Conventional approaches to cryopreservation that provide the cornerstone of isolated cell storage have not been successfully extrapolated to more complex natural, or engineered, multicellular tissues. Tissues are much more than simple aggregates of various cell types; they have a highly organized, often complex, structure that influences their response to freezing and thawing. Cryopreservation is a complex process of coupled heat and mass transfer generally executed under non-equilibrium conditions. Advances in the field were modest until the cryoprotective properties of glycerol and dimethyl sulfoxide (DMSO) were discovered in the mid 1900's.1,2 Many other cryoprotective agents (CPAs) have since been identified. Combinations of CPAs may result in additive or synergistic enhancement of cell survival by minimization of intracellular ice during freezing.3 
Restriction of the amount and size of extracellular ice crystal formation during cryopreservation can be achieved by using high concentrations of CPAs that promote amorphous solidification, known as vitrification, rather than crystallization.4 Vitrification is a relatively well understood physical process, but its application to the preservation of biological systems has not been without problems, since the high concentrations of CPAs necessary to facilitate vitrification are potentially toxic. To limit toxic effects, it is necessary to use the least toxic CPAs at the lowest concentrations that will still permit glass formation (at cooling rates that are practical for bulky mammalian tissues).4 Comparison of the effects of vitrification and conventional frozen cryopreservation upon venous contractility demonstrate that vitrification is superior to conventional cryopreservation methods for tissues.4 However, both vitrification and conventional freezing methods have their place in the cryopreservation of cellular materials.
However, both conventional freezing and vitrification approaches to preservation have limitations. First, both of these technologies require low temperature storage and transportation conditions. Neither can be stored above their glass transition temperature for very long without significant risk of product damage due to ice formation and growth. Both technologies require competent technical support during the rewarming and CPA elution phase prior to product utilization. This is possible in a high technology surgical operating theater but not in a doctor's outpatient office or in third world environments. In contrast, theoretically, a dry product would have none of these issues because it should be stable at room temperature and rehydration should be feasible in a sterile packaging system.
Drying and vitrification have previously been combined for matrix preservation of cardiovascular and skin tissues, but not for live cell preservation in tissues or engineered products. Nature, however, has developed a wide variety of organisms and animals that tolerate dehydration stress by a spectrum of physiological and genetic adaptation mechanisms. Among these adaptive processes, the accumulation of large amounts of disaccharides, especially trehalose and sucrose, is especially noteworthy in almost all anhydrobiotic organisms including plant seeds, bacteria, insects, yeast, brine shrimp, fungi and their spores, cysts of certain crustaceans, and some soil-dwelling animals.5-7 The protective effects of trehalose and sucrose may be classified under two general mechanisms: (1) “the water replacement hypothesis” or stabilization of biological membranes and proteins by direct interaction of sugars with polar residues through hydrogen bonding, and (2) stable glass formation (vitrification) by sugars in the dry state.
The stabilizing effect of these sugars has also been shown in a number of model systems, including liposomes, membranes, viral particles, and proteins during dry storage at ambient temperatures.8-10 On the other hand, the use of these sugars in mammalian cells has been somewhat limited, mainly because mammalian cell membranes are believed to be impermeable to disaccharides or larger sugars.11 For sugars to be effective, it is believed that they need to be present both on the inside and the outside of the cell membrane. Several methods have been developed for loading of sugars in living cells. Recently, a novel, genetically-modified, metal-actuated switchable membrane pore has been used to reversibly permeabilize mammalian cells to sugars with significant post-cryopreservation and, to lesser extent, drying cell survival.12 Other permeation technologies that have been considered for placing sugars in cells include use of microinjection and thermal shock. The expression of sucrose and trehalose synthase genes and transporters has also been considered as means for delivery of sugars into cells. Introduction of trehalose into human pancreatic islet cells during a cell membrane thermotropic lipid-phase transition, prior to freezing and in the presence of a mixture of 2M DMSO and trehalose, resulted in previously unattainable cell survival rates.13 This method depends upon suspension of cells in a trehalose solution and either cooling or warming the solution through the thermotropic transition of the cells.14 Human fibroblast transfection with E. coli genes expressing trehalose resulted in retention of viability after drying for up to five days.15 